The extracytoplasmic function (ECF) sigma factor sR is a global regulator of redox homeostasis in... more The extracytoplasmic function (ECF) sigma factor sR is a global regulator of redox homeostasis in the antibiotic-producing bacterium Streptomyces coelicolor, with a similar role in other actinomycetes such as Mycobacterium tuberculosis. Normally maintained in an inactive state by its bound anti-sigma factor RsrA, sR dissociates in response to intracellular disulphide-stress to direct core RNA polymerase to transcribe genes, such as trxBA and trxC that encode the enzymes of the thioredoxin disulphide reductase pathway, that re-establish redox homeostasis. Little is known about where RsrA binds on sR or how it suppresses sR-dependent transcriptional activity. Using a combination of proteolysis, surface-enhanced laser desorption ionisation mass spectrometry and pull-down assays we identify an N-terminal,,10 kDa domain (sRN) that encompasses region 2 of sR that represents the major RsrA binding site. We show that sRN inhibits transcription by an unrelated sigma factor and that this inhi...
Novel bacterial type II topoisomerase inhibitors (NBTIs) stabilize single-strand DNA cleavage bre... more Novel bacterial type II topoisomerase inhibitors (NBTIs) stabilize single-strand DNA cleavage breaks by DNA gyrase but their exact mechanism of action has remained hypothetical until now. We have designed a small library of NBTIs with an improved DNA gyrase-binding moiety resulting in low nanomolar inhibition and very potent antibacterial activity. They stabilize single-stranded cleavage complexes and, importantly, we have obtained the crystal structure where an NBTI binds gyrase–DNA in a single conformation lacking apparent static disorder. This directly proves the previously postulated NBTI mechanism of action and shows that they stabilize single-strand cleavage through asymmetric intercalation with a shift of the scissile phosphate. This crystal stucture shows that the chlorine forms a halogen bond with the backbone carbonyls of the two symmetry-related Ala68 residues. To the best of our knowledge, such a so-called symmetrical bifurcated halogen bond has not been identified in a ...
Transcription in HeLa cell extracts in vitro was stimulated 8- to 10-fold by a recombinant chimer... more Transcription in HeLa cell extracts in vitro was stimulated 8- to 10-fold by a recombinant chimera, GAL-TEF-1, consisting of the DNA-binding domain of GAL4 and the activation function of the HeLa cell activator TEF-1. In contrast, only a 2- to 3-fold stimulation was obtained with GAL-TEF-1 in extracts from BJA-B lymphoid cells. Stimulation by GAL-TEF-1 in BJA-B extracts was dramatically increased by the addition of immunopurified HeLa cell TFIID, suggesting that BJA-B TFIID lacks or contains lower quantities of a TATA-binding-protein-associated factor(s) required for the activity of the TEF-1 activation function. However, chromatography, immunopurification, and transcriptional reconstitution experiments indicated that BJA-B extracts did not lack the previously identified TATA-binding-protein-associated factors required for TEF-1 activity but rather contained a negatively acting factor(s) which inhibited transactivation by GAL-TEF-1. These results indicate that the relative lack of a...
Protein-Dye Interactions: Developments and Applications, 1989
Downstream processing refers to all the technologies that are responsible for the production of p... more Downstream processing refers to all the technologies that are responsible for the production of pure products after fermentation. Therefore, if one were to produce, for example, a protein, one would start with an appropriate bio-reactor containing native or engineered cells. The first step would be to separate the cells from the broth; if the product is an intra-cellular one, one would subsequently disrupt the cells to release the intra-cellular products and then embark on a clarification process to obtain a clear extract containing the protein of interest. The next step is to apply a whole series of high resolution purification techniques, particularly chromatographic steps, prior to subsequently ending up with pure protein. Therefore, downstream processing entails the execution of primary recovery stages followed by a series of high-resolution steps where we add value to the final product and then hopefully end up with pure homogeneous protein. Special interest is focused in this report on the high resolution stages of the process leading to pure product and particularly those steps involving the most refined version of chromatography, affinity chromatography (1). The technique of affinity chromatography exploits small ligands which bind specifically and reversibly to the protein of interest. The appropriate small ligand is covalently attached to a suitable solid support matrix in such a way that we can establish that as a column.
Methods in molecular biology (Clifton, N.J.), 2010
We have developed a rapid, high-throughput assay for measuring the catalytic activity (DNA superc... more We have developed a rapid, high-throughput assay for measuring the catalytic activity (DNA supercoiling or relaxation) of topoisomerase enzymes that is also capable of monitoring the activity of other enzymes that alter the topology of DNA. The assay utilises intermolecular triplex formation to resolve supercoiled and relaxed forms of DNA, the principle being the greater efficiency of a negatively supercoiled plasmid to form an intermolecular triplex with an immobilised oligonucleotide than the relaxed form. The assay provides a number of advantages over the standard gel-based methods, including greater speed of analysis, reduced sample handling, better quantitation and improved reliability and accuracy of output data. The assay is performed in microtitre plates and can be adapted to high-throughput screening of libraries of potential inhibitors of topoisomerases including bacterial DNA gyrase.
Affinity chromatography has been extensively refined over the past few years to meet the more str... more Affinity chromatography has been extensively refined over the past few years to meet the more stringent criieria being placed on recombinant proteins as therapeutic products. New developments in the design of selective and stable ligands for affinity chromatography are establishing the technique as a routine tool in processscale protein purification. Exploitation of sophisticated molecular modelling kchniques in conjunction with binding and crystallographic studies has permitted
Proteins: Structure, Function, and Bioinformatics, 2006
Introduction. The RAD gene of Antirrhinum majus encodes a single-repeat MYB transcription factor ... more Introduction. The RAD gene of Antirrhinum majus encodes a single-repeat MYB transcription factor that is implicated in the control of floral asymmetry. Herein we present the crystal structure of the MYB domain of the RAD protein determined using the anomalous signal of iodide and refined to a resolution of 1.9 A ˚. This consti-
Proceedings of the National Academy of Sciences, 1991
We provide biochemical evidence for the existence of a transcriptional intermediary factor (TIF) ... more We provide biochemical evidence for the existence of a transcriptional intermediary factor (TIF) in HeLa whole-cell extracts (WCE) that is distinct from the basic transcription factors and that is required for transcriptional stimulation by the chimeric acidic activator GAL-VP16. We have fractionated HeLa WCE by heparin-agarose chromatography. Of transcriptionally active fractions eluting in a step between 0.24 and 0.6 M KCl, the initial fractions are refractory to GAL-VP16 stimulation, whereas subsequent fractions are strongly stimulated by the activator. Aliquots of GAL-VP16-responsive fractions efficiently complement refractory fractions for transcriptional stimulation. Aliquots of responsive fractions are also far more efficient than those of refractory fractions in overcoming transcriptional inhibition that is brought about by high concentrations of GAL-VP16. Experiments performed with heat-treated WCE support the idea that HeLa cells contain a TIF that is essential for GAL-VP1...
We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topo... more We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled plasmids form intermolecular triplexes more efficiently than when they are relaxed. Two assays are presented, one using capture of a plasmid containing a single triplexforming sequence by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by staining with a DNA-specific fluorescent dye. The other uses capture of a plasmid containing two triplex-forming sequences by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by a second oligonucleotide that is radiolabelled. The assays are shown to be appropriate for assaying DNA supercoiling by Escherichia coli DNA gyrase and DNA relaxation by eukaryotic topoisomerases I and II, and E.coli topoisomerase IV. The assays are readily adaptable to other enzymes that change DNA supercoiling (e.g. restriction enzymes) and are suitable for use in a high-throughput format.
Mineral sulfide concentrate oxidation by thermoacidophilic archaea was reported in the 1970s with... more Mineral sulfide concentrate oxidation by thermoacidophilic archaea was reported in the 1970s with a culture which was described as ''possibly related to Sulfolobus'' but which contained the species later named as Acidianus brierleyi. In the following two decades, the mineral sulfide-oxidizing capacity of Sulfolobus metallicus became the subject of many studies partly because it appeared to tolerate higher concentrations of copper and solids in bioreactors than those tolerated by the strains of Acidianus and Metallosphaera that were available. However, some bioreactor pilot plant developments followed with mixed cultures dominated by A. brierleyi at 70 °C. At higher temperatures, pilot and industrial demonstration plants used mixed cultures dominated by poorly characterized novel species. Most of these novel species will require classification in novel genera to reflect their phylogenetic separation from previously named thermoacidophilic archaea.
A number of new cationic ligands have been designed and synthesized for the selective resolution ... more A number of new cationic ligands have been designed and synthesized for the selective resolution and purification of the trypszin-like proteases. A series of ligands based on 4-[2'-methyl-4'-(2",4"-dichloro-l",~,S-triazin-6ylamino) phenylazo]benzamidine were able to bind to trypsin and the trypsin-like proteases, thrombin and urokinase, but bound pancreatic kallikrein only weakly. Ligands possessing a second cationic group (either 4aminophenyltrimethylammonium or 4-aminobenzamidine) substituted onto the triazine ring displayed higher affinities than the parent compound for trypsin in solution but bound the enzyme weakly or not at all after immobilization. In contrast, these bis-cationic ligands bound pancreatic kallikrein in solution and following immobilization. The presence of the second cationic group was crucial, since its replacement by neutral or anionic groups led to loss of affinity for pancreatic kallikrein. One of the bis-cationic ligands was used to purify pancreatic kallikrein 9.5-fold from a crude pancreatic extract in 79% yield, to generate a product 99.9% free of contaminating trypsin activity.
A number of ligands for the selective purification by affinity chromatography of the trypsin-like... more A number of ligands for the selective purification by affinity chromatography of the trypsin-like protease, porcine pancreatic kallikrein, were designed de nouo by computer-aided molecular design. The ligands were designed to mimic the side-chains of a number of arginyl dipeptides and included a benzamidine moiety substituted on a triazine ring. The ligands displayed inhibitory activities against pancreatic kallikrein which mirrored the specificity constants of the dipeptides they were designed to mimic. The ligand with the highest affinity for the enzyme, an analogue of a Phe-Arg dipeptide, when immobilized to Sepharose CL-4B via a hexamethylene spacer arm, purified pancreatic kallikrein 110-fold in one step from a crude pancreatic acetone extract.
The extracytoplasmic function (ECF) sigma factor s R is a global regulator of redox homeostasis i... more The extracytoplasmic function (ECF) sigma factor s R is a global regulator of redox homeostasis in the antibiotic-producing bacterium Streptomyces coelicolor, with a similar role in other actinomycetes such as Mycobacterium tuberculosis. Normally maintained in an inactive state by its bound antisigma factor RsrA, s R dissociates in response to intracellular disulphidestress to direct core RNA polymerase to transcribe genes, such as trxBA and trxC that encode the enzymes of the thioredoxin disulphide reductase pathway, that re-establish redox homeostasis. Little is known about where RsrA binds on s R or how it suppresses s R -dependent transcriptional activity. Using a combination of proteolysis, surface-enhanced laser desorption ionisation mass spectrometry and pull-down assays we identify an N-terminal, , 10 kDa domain (s RN ) that encompasses region 2 of s R that represents the major RsrA binding site. We show that s RN inhibits transcription by an unrelated sigma factor and that this inhibition is relieved by RsrA binding, reaffirming that region 2 is involved in binding to core RNA polymerase but also demonstrating that the likely mechanism by which RsrA inhibits s R activity is by blocking this association. We also report the 2.4 A ˚resolution crystal structure of s RN that reveals extensive structural conservation with the equivalent region of s 70 from Escherichia coli as well as with the cyclin-box, a domain-fold found in the eukaryotic proteins TFIIB and cyclin A. s RN has a propensity to aggregate, due to steric complementarity of oppositely charged surfaces on the domain, but this is inhibited by RsrA, an observation that suggests a possible mode of action for RsrA which we compare to other well-studied sigma factor-anti-sigma factor systems.
The production of a 25 kDa protein was greatly increased when a sulfur-and ferrous iron-oxidizing... more The production of a 25 kDa protein was greatly increased when a sulfur-and ferrous iron-oxidizing species of Su(fu/ohu.s was switched from growth on tetrathionate to growth on ferrous iron. The gene encoding the protein was cloned and sequenced. The predicted amino sequence showed significant similarity to those of the alkyl hydroperoxide reductase/thiol specific anti-oxidant family of proteins that appear to be involved in responses to certain types of oxidative stress.
DNA was extracted from water and sediment samples taken from acidic, geothermal pools on the Cari... more DNA was extracted from water and sediment samples taken from acidic, geothermal pools on the Caribbean island of Montserrat. 16S rRNA genes were amplified by PCR, cloned, sequenced, and examined to indicate some of the organisms that might be significant components of the in situ microbiota. A clone bank representing the lowest temperature pool that was sampled (33 degrees C) was dominated by genes corresponding to two types of acidophiles: Acidiphilium-like mesophilic heterotrophs and thermotolerant Acidithiobacillus caldus. Three clone types with origins in low- and moderate- (48 degrees C) temperature pools corresponded to bacteria that could be involved in metabolism of sulfur compounds: the aerobic A. caldus and putative anaerobic, moderately thermophilic, sulfur-reducing bacteria (from an undescribed genus and from the Desulfurella group). A higher-temperature sample indicated the presence of a Ferroplasma-like organism, distinct from the other strains of these recently recognized acidophilic, iron-oxidizing members of the Euryarchaeota. Acidophilic Archaea from undescribed genera related to Sulfolobus and Acidianus were predicted to dominate the indigenous acidophilic archaeal population at the highest temperatures.
Soluble methane monooxygenase (sMMO) of Methylosinus trichosporium OB3b is a three-component oxyg... more Soluble methane monooxygenase (sMMO) of Methylosinus trichosporium OB3b is a three-component oxygenase that catalyses the O 2 - and NAD(P)H-dependent oxygenation of methane and numerous other substrates. Despite substantial interest in the use of genetic techniques to study the mechanism of sMMO and manipulate its substrate specificity, directed mutagenesis of active-site residues was previously impossible because no suitable heterologous expression system had been found for expression in a highly active form of the hydroxylase component, which is an (αβγ) 2 complex containing the binuclear iron active site. A homologous expression system that enabled the expression of recombinant wild-type sMMO in a derivative of M . trichosporium OB3b from which the chromosomal copy of the sMMO-encoding operon had been partially deleted was previously reported. Here we report substantial development of this method to produce a system for the facile construction and expression of mutants of the hyd...
The extracytoplasmic function (ECF) sigma factor sR is a global regulator of redox homeostasis in... more The extracytoplasmic function (ECF) sigma factor sR is a global regulator of redox homeostasis in the antibiotic-producing bacterium Streptomyces coelicolor, with a similar role in other actinomycetes such as Mycobacterium tuberculosis. Normally maintained in an inactive state by its bound anti-sigma factor RsrA, sR dissociates in response to intracellular disulphide-stress to direct core RNA polymerase to transcribe genes, such as trxBA and trxC that encode the enzymes of the thioredoxin disulphide reductase pathway, that re-establish redox homeostasis. Little is known about where RsrA binds on sR or how it suppresses sR-dependent transcriptional activity. Using a combination of proteolysis, surface-enhanced laser desorption ionisation mass spectrometry and pull-down assays we identify an N-terminal,,10 kDa domain (sRN) that encompasses region 2 of sR that represents the major RsrA binding site. We show that sRN inhibits transcription by an unrelated sigma factor and that this inhi...
Novel bacterial type II topoisomerase inhibitors (NBTIs) stabilize single-strand DNA cleavage bre... more Novel bacterial type II topoisomerase inhibitors (NBTIs) stabilize single-strand DNA cleavage breaks by DNA gyrase but their exact mechanism of action has remained hypothetical until now. We have designed a small library of NBTIs with an improved DNA gyrase-binding moiety resulting in low nanomolar inhibition and very potent antibacterial activity. They stabilize single-stranded cleavage complexes and, importantly, we have obtained the crystal structure where an NBTI binds gyrase–DNA in a single conformation lacking apparent static disorder. This directly proves the previously postulated NBTI mechanism of action and shows that they stabilize single-strand cleavage through asymmetric intercalation with a shift of the scissile phosphate. This crystal stucture shows that the chlorine forms a halogen bond with the backbone carbonyls of the two symmetry-related Ala68 residues. To the best of our knowledge, such a so-called symmetrical bifurcated halogen bond has not been identified in a ...
Transcription in HeLa cell extracts in vitro was stimulated 8- to 10-fold by a recombinant chimer... more Transcription in HeLa cell extracts in vitro was stimulated 8- to 10-fold by a recombinant chimera, GAL-TEF-1, consisting of the DNA-binding domain of GAL4 and the activation function of the HeLa cell activator TEF-1. In contrast, only a 2- to 3-fold stimulation was obtained with GAL-TEF-1 in extracts from BJA-B lymphoid cells. Stimulation by GAL-TEF-1 in BJA-B extracts was dramatically increased by the addition of immunopurified HeLa cell TFIID, suggesting that BJA-B TFIID lacks or contains lower quantities of a TATA-binding-protein-associated factor(s) required for the activity of the TEF-1 activation function. However, chromatography, immunopurification, and transcriptional reconstitution experiments indicated that BJA-B extracts did not lack the previously identified TATA-binding-protein-associated factors required for TEF-1 activity but rather contained a negatively acting factor(s) which inhibited transactivation by GAL-TEF-1. These results indicate that the relative lack of a...
Protein-Dye Interactions: Developments and Applications, 1989
Downstream processing refers to all the technologies that are responsible for the production of p... more Downstream processing refers to all the technologies that are responsible for the production of pure products after fermentation. Therefore, if one were to produce, for example, a protein, one would start with an appropriate bio-reactor containing native or engineered cells. The first step would be to separate the cells from the broth; if the product is an intra-cellular one, one would subsequently disrupt the cells to release the intra-cellular products and then embark on a clarification process to obtain a clear extract containing the protein of interest. The next step is to apply a whole series of high resolution purification techniques, particularly chromatographic steps, prior to subsequently ending up with pure protein. Therefore, downstream processing entails the execution of primary recovery stages followed by a series of high-resolution steps where we add value to the final product and then hopefully end up with pure homogeneous protein. Special interest is focused in this report on the high resolution stages of the process leading to pure product and particularly those steps involving the most refined version of chromatography, affinity chromatography (1). The technique of affinity chromatography exploits small ligands which bind specifically and reversibly to the protein of interest. The appropriate small ligand is covalently attached to a suitable solid support matrix in such a way that we can establish that as a column.
Methods in molecular biology (Clifton, N.J.), 2010
We have developed a rapid, high-throughput assay for measuring the catalytic activity (DNA superc... more We have developed a rapid, high-throughput assay for measuring the catalytic activity (DNA supercoiling or relaxation) of topoisomerase enzymes that is also capable of monitoring the activity of other enzymes that alter the topology of DNA. The assay utilises intermolecular triplex formation to resolve supercoiled and relaxed forms of DNA, the principle being the greater efficiency of a negatively supercoiled plasmid to form an intermolecular triplex with an immobilised oligonucleotide than the relaxed form. The assay provides a number of advantages over the standard gel-based methods, including greater speed of analysis, reduced sample handling, better quantitation and improved reliability and accuracy of output data. The assay is performed in microtitre plates and can be adapted to high-throughput screening of libraries of potential inhibitors of topoisomerases including bacterial DNA gyrase.
Affinity chromatography has been extensively refined over the past few years to meet the more str... more Affinity chromatography has been extensively refined over the past few years to meet the more stringent criieria being placed on recombinant proteins as therapeutic products. New developments in the design of selective and stable ligands for affinity chromatography are establishing the technique as a routine tool in processscale protein purification. Exploitation of sophisticated molecular modelling kchniques in conjunction with binding and crystallographic studies has permitted
Proteins: Structure, Function, and Bioinformatics, 2006
Introduction. The RAD gene of Antirrhinum majus encodes a single-repeat MYB transcription factor ... more Introduction. The RAD gene of Antirrhinum majus encodes a single-repeat MYB transcription factor that is implicated in the control of floral asymmetry. Herein we present the crystal structure of the MYB domain of the RAD protein determined using the anomalous signal of iodide and refined to a resolution of 1.9 A ˚. This consti-
Proceedings of the National Academy of Sciences, 1991
We provide biochemical evidence for the existence of a transcriptional intermediary factor (TIF) ... more We provide biochemical evidence for the existence of a transcriptional intermediary factor (TIF) in HeLa whole-cell extracts (WCE) that is distinct from the basic transcription factors and that is required for transcriptional stimulation by the chimeric acidic activator GAL-VP16. We have fractionated HeLa WCE by heparin-agarose chromatography. Of transcriptionally active fractions eluting in a step between 0.24 and 0.6 M KCl, the initial fractions are refractory to GAL-VP16 stimulation, whereas subsequent fractions are strongly stimulated by the activator. Aliquots of GAL-VP16-responsive fractions efficiently complement refractory fractions for transcriptional stimulation. Aliquots of responsive fractions are also far more efficient than those of refractory fractions in overcoming transcriptional inhibition that is brought about by high concentrations of GAL-VP16. Experiments performed with heat-treated WCE support the idea that HeLa cells contain a TIF that is essential for GAL-VP1...
We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topo... more We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled plasmids form intermolecular triplexes more efficiently than when they are relaxed. Two assays are presented, one using capture of a plasmid containing a single triplexforming sequence by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by staining with a DNA-specific fluorescent dye. The other uses capture of a plasmid containing two triplex-forming sequences by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by a second oligonucleotide that is radiolabelled. The assays are shown to be appropriate for assaying DNA supercoiling by Escherichia coli DNA gyrase and DNA relaxation by eukaryotic topoisomerases I and II, and E.coli topoisomerase IV. The assays are readily adaptable to other enzymes that change DNA supercoiling (e.g. restriction enzymes) and are suitable for use in a high-throughput format.
Mineral sulfide concentrate oxidation by thermoacidophilic archaea was reported in the 1970s with... more Mineral sulfide concentrate oxidation by thermoacidophilic archaea was reported in the 1970s with a culture which was described as ''possibly related to Sulfolobus'' but which contained the species later named as Acidianus brierleyi. In the following two decades, the mineral sulfide-oxidizing capacity of Sulfolobus metallicus became the subject of many studies partly because it appeared to tolerate higher concentrations of copper and solids in bioreactors than those tolerated by the strains of Acidianus and Metallosphaera that were available. However, some bioreactor pilot plant developments followed with mixed cultures dominated by A. brierleyi at 70 °C. At higher temperatures, pilot and industrial demonstration plants used mixed cultures dominated by poorly characterized novel species. Most of these novel species will require classification in novel genera to reflect their phylogenetic separation from previously named thermoacidophilic archaea.
A number of new cationic ligands have been designed and synthesized for the selective resolution ... more A number of new cationic ligands have been designed and synthesized for the selective resolution and purification of the trypszin-like proteases. A series of ligands based on 4-[2'-methyl-4'-(2",4"-dichloro-l",~,S-triazin-6ylamino) phenylazo]benzamidine were able to bind to trypsin and the trypsin-like proteases, thrombin and urokinase, but bound pancreatic kallikrein only weakly. Ligands possessing a second cationic group (either 4aminophenyltrimethylammonium or 4-aminobenzamidine) substituted onto the triazine ring displayed higher affinities than the parent compound for trypsin in solution but bound the enzyme weakly or not at all after immobilization. In contrast, these bis-cationic ligands bound pancreatic kallikrein in solution and following immobilization. The presence of the second cationic group was crucial, since its replacement by neutral or anionic groups led to loss of affinity for pancreatic kallikrein. One of the bis-cationic ligands was used to purify pancreatic kallikrein 9.5-fold from a crude pancreatic extract in 79% yield, to generate a product 99.9% free of contaminating trypsin activity.
A number of ligands for the selective purification by affinity chromatography of the trypsin-like... more A number of ligands for the selective purification by affinity chromatography of the trypsin-like protease, porcine pancreatic kallikrein, were designed de nouo by computer-aided molecular design. The ligands were designed to mimic the side-chains of a number of arginyl dipeptides and included a benzamidine moiety substituted on a triazine ring. The ligands displayed inhibitory activities against pancreatic kallikrein which mirrored the specificity constants of the dipeptides they were designed to mimic. The ligand with the highest affinity for the enzyme, an analogue of a Phe-Arg dipeptide, when immobilized to Sepharose CL-4B via a hexamethylene spacer arm, purified pancreatic kallikrein 110-fold in one step from a crude pancreatic acetone extract.
The extracytoplasmic function (ECF) sigma factor s R is a global regulator of redox homeostasis i... more The extracytoplasmic function (ECF) sigma factor s R is a global regulator of redox homeostasis in the antibiotic-producing bacterium Streptomyces coelicolor, with a similar role in other actinomycetes such as Mycobacterium tuberculosis. Normally maintained in an inactive state by its bound antisigma factor RsrA, s R dissociates in response to intracellular disulphidestress to direct core RNA polymerase to transcribe genes, such as trxBA and trxC that encode the enzymes of the thioredoxin disulphide reductase pathway, that re-establish redox homeostasis. Little is known about where RsrA binds on s R or how it suppresses s R -dependent transcriptional activity. Using a combination of proteolysis, surface-enhanced laser desorption ionisation mass spectrometry and pull-down assays we identify an N-terminal, , 10 kDa domain (s RN ) that encompasses region 2 of s R that represents the major RsrA binding site. We show that s RN inhibits transcription by an unrelated sigma factor and that this inhibition is relieved by RsrA binding, reaffirming that region 2 is involved in binding to core RNA polymerase but also demonstrating that the likely mechanism by which RsrA inhibits s R activity is by blocking this association. We also report the 2.4 A ˚resolution crystal structure of s RN that reveals extensive structural conservation with the equivalent region of s 70 from Escherichia coli as well as with the cyclin-box, a domain-fold found in the eukaryotic proteins TFIIB and cyclin A. s RN has a propensity to aggregate, due to steric complementarity of oppositely charged surfaces on the domain, but this is inhibited by RsrA, an observation that suggests a possible mode of action for RsrA which we compare to other well-studied sigma factor-anti-sigma factor systems.
The production of a 25 kDa protein was greatly increased when a sulfur-and ferrous iron-oxidizing... more The production of a 25 kDa protein was greatly increased when a sulfur-and ferrous iron-oxidizing species of Su(fu/ohu.s was switched from growth on tetrathionate to growth on ferrous iron. The gene encoding the protein was cloned and sequenced. The predicted amino sequence showed significant similarity to those of the alkyl hydroperoxide reductase/thiol specific anti-oxidant family of proteins that appear to be involved in responses to certain types of oxidative stress.
DNA was extracted from water and sediment samples taken from acidic, geothermal pools on the Cari... more DNA was extracted from water and sediment samples taken from acidic, geothermal pools on the Caribbean island of Montserrat. 16S rRNA genes were amplified by PCR, cloned, sequenced, and examined to indicate some of the organisms that might be significant components of the in situ microbiota. A clone bank representing the lowest temperature pool that was sampled (33 degrees C) was dominated by genes corresponding to two types of acidophiles: Acidiphilium-like mesophilic heterotrophs and thermotolerant Acidithiobacillus caldus. Three clone types with origins in low- and moderate- (48 degrees C) temperature pools corresponded to bacteria that could be involved in metabolism of sulfur compounds: the aerobic A. caldus and putative anaerobic, moderately thermophilic, sulfur-reducing bacteria (from an undescribed genus and from the Desulfurella group). A higher-temperature sample indicated the presence of a Ferroplasma-like organism, distinct from the other strains of these recently recognized acidophilic, iron-oxidizing members of the Euryarchaeota. Acidophilic Archaea from undescribed genera related to Sulfolobus and Acidianus were predicted to dominate the indigenous acidophilic archaeal population at the highest temperatures.
Soluble methane monooxygenase (sMMO) of Methylosinus trichosporium OB3b is a three-component oxyg... more Soluble methane monooxygenase (sMMO) of Methylosinus trichosporium OB3b is a three-component oxygenase that catalyses the O 2 - and NAD(P)H-dependent oxygenation of methane and numerous other substrates. Despite substantial interest in the use of genetic techniques to study the mechanism of sMMO and manipulate its substrate specificity, directed mutagenesis of active-site residues was previously impossible because no suitable heterologous expression system had been found for expression in a highly active form of the hydroxylase component, which is an (αβγ) 2 complex containing the binuclear iron active site. A homologous expression system that enabled the expression of recombinant wild-type sMMO in a derivative of M . trichosporium OB3b from which the chromosomal copy of the sMMO-encoding operon had been partially deleted was previously reported. Here we report substantial development of this method to produce a system for the facile construction and expression of mutants of the hyd...
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